Atherosclerosis is a chronic inflammatory disease that is promoted by the consumption of dietary fat and cholesterol. The extent to which atherosclerosis progresses to cause coronary occlusion and/or death is mediated by the presence of plasma apoA-l, the main protein constituent of high density lipoproteins (HDL). The role of HDL apoA-l in whole body cholesterol homeostasis has been extensively investigated and its role is to direct cholesterol from the periphery to the liver for excretion. Despite intensive investigations, major questions remain regarding the mechanism by which apoA-l regulates cellular cholesterol levels and how the apoprotien's unique structural features facilitate cholesterol transport. To more completely understand the structural basis for apoA-l's role in cholesterol transport, we will carry out three specific aims that will clarify the structural reorganization necessary for the formation of nascent HDL via ABCA1. In Aim 1 we propose to address the role of apoA-l in cholesterol homeostasis by determining the conformation of apoA-l on four different sized subclasses of ABCA1 generated nascent HDL. Also as part of Aim 1, we will examine the 'unfolding'steps'through which lipid-free apoA-l acquires lipid from ABCA1. To do this we will construct a series of 'tethered'disulfide apoA-l mutants that will prevent key intermediate unfolding steps through which the 4-helix bundle must transition as it organizes and binds phospholipid and cholesterol. In the Aim 2, we will investigate the mechanistic basis for the dominant negative repression of wild-type apoA-l HDL, as observed in humans who carry this mutation, by investigating the lipidation of the helix 6 mutant, L159R apoA-l by ABCA1. Our preliminary studies suggest that this single amino acid substitution mutant, L159R apoA-l, competes with wild-type apoA-l for phospholipid and cholesterol, resulting in a reduction in the overall lipidation of wild-type apoA-l by ABCA1 in a model cell culture system. In Aim 3, we plan to determine whether the dominant negative phenotype associated with the L159R apoA-l mutant is pro- or antiatherogenic in hyperlipidemic mice. Using transgenic mice that express wild-type or L159R apoA-l, we will determine the extent to which the mutant apoA-l protects against the development of atherosclerosis, as well as investigating the in vivo basis for the low concentration of L159R apoA-l in plasma as it relates to the dominant negative phenotype.